Method of treating bone disease with pyridine, carboxamide and carboxylic derivatives

ABSTRACT

Treatment of osteopathic comprising administering as an active ingredient a compound represented by the following formula (I), (II) or (III): ##STR1##

The present invention relates to an anti-osteopathic composition usefulfor therapeutic and prophylactic treatment of osteopathic. Morespecifically, the present invention relates to an anti-osteopathiccomposition comprising an aromatic carboxylic acid or its derivative asan active ingredient. The anti-osteopathic composition can be used for,for example, a therapeutic and prophylactic treatment of osteodysbolismor a therapeutic and prophylactic treatment of fractures.

Metabolism of bones is considered to be achieved by well balancedrepetitions of bone resorption by osteoclasts and osteogenesis byosteoblasts. Dynamic equilibrium is maintained between such generationprocess and resorption process in healthy adults and, as a result,weights and structures of their bones are maintained. It is suggestedthat main cause of various osteodysbolisms, including osteoporosis as antypical example, is an abnormal mutual cooperation of these twoprocesses.

Osteoporosis is a disease in which reductions of bone densities and boneamounts are brought about as a result of excess bone resorptions whichare induced by a lost of the aforementioned balance with an increasedbone resorption. This disease outbreaks in adults and is frequentlyobserved particularly in women of middle and advanced age. Patients ofthis disease are susceptible to fractures because of the reductions ofbone density and amount, and some patients fall into serious conditions,such as conditions where they are entirely in beds. Therefore,development of therapeutic drugs effective for osteoporosis is beinghighly desired.

As medicaments for the treatment of such osteopathic, compositions suchas, for example, those comprising activated-form vitamin D₃, calcitonin,bis-phosphonic acid, estrogen, ipriflavone, and calcium are used. Mostof these pharmaceutical compositions are reported to have inhibitoryactivity on bone resorption or the like. However, it has not beenclearly demonstrated that these compositions have enhancing activitieson osteogenesis.

Vitamin A acid (retinoic acid), an oxidative metabolite of vitamin A(retinol), has been reported to have therapeutic efficacy on certaintypes of leukemia, skin cancer and intractable skin diseases, inaddition to its pharmacological activities relating to life sustainment,embryogenesis and growth of animals. Among retinoids (a generic term forcompounds exhibiting retinoic acid-type biological activities) havingvarious pharmacological activities, isotretinoin and etretinate having apolyene structure similar to that of retinoic acid are practically usedas clinical medicaments. They are mainly used as medicaments for thetherapeutic treatment of intractable skin diseases.

So far, some experimental results in vitro with respect to an activityof retinoic acid on osteocytes have been reported (R. P. J. Oneill etal., Bone, Vol. 13, pp. 29-47, 1992). However, effects of retinoic acidon osteocytes have been remained unclear because contrary results havebeen also reported. Furthermore, etretinate, one of synthetic retinoids,has been known to induce a clinical side effect of hyperostosis (Methodsin Enzymology, Vol. 190, pp. 291-304, 1990, Academic Press). U.S. Pat.No. 5,070,108 discloses a method of using etretinate for the treatmentof osteoporosis. However, retinoic acid, retinol, retinal, etretinate,isotretinoin and the like are not sufficiently effective as medicamentfor the treatment of osteopathic, in particular, osteoporosis, sincethey are unstable due to their polyene structures, and moreover, theyhave various kinds of pharmacological activities.

Therefore, an object of the present invention is to provide retinoidcompounds which successfully enhance osteogenesis at low concentrationswith high specificity for osteogenesis enhancing activity and arechemically stable. The inventors of the present invention conductedvarious studies to achieve the foregoing object, and as a result, theyfound that aromatic carboxylic acids and their derivatives set out beloware effective for therapeutic and prophylactic treatment of osteopathia.The present invention was achieved on the basis of these findings. Inthe specification, the term "osteopathia" means all sorts of diseasesrelating to bones such as, for example, bone metabolic disorders andbone fractures. The anti-osteopathic composition of the presentinvention can be used for therapeutic and prophylactic treatment ofosteopathia such as, for example, osteoporosis and bone fracture.

The present invention thus provides an anti-osteopathic compositioncomprising as an active ingredient a compound represented by thefollowing formula (I): ##STR2## wherein R₁, R₂, R₃, R₄ and R₅independently represent a hydrogen atom, a lower or middle alkyl group,or a lower or middle alkoxy group, with the proviso that all of R₁, R₂,R₃, R₄ and R₅ do not simultaneously represent hydrogen atoms, any twoadjacent groups selected from these groups may combine to form a 5- or6-membered cycloalkyl ring together with carbon atoms substituted bythose groups and the cycloalkyl ring may further be substituted with oneor more lower alkyl groups; R₆ represents a hydroxyl group, a loweralkoxy group, or --NR₇ R₈ group in which R₇ and R₈ independentlyrepresent a hydrogen atom or a lower alkyl group; X represents a radicalselected from the group consisting of the radicals represented by thefollowing formulas: ##STR3## wherein R₉ and R₁₀ independently representsa hydrogen atom or a lower alkyl group and; Y represents a radicalselected from the group consisting of the radicals represented by thefollowing formulas: ##STR4## wherein W represents a hydrogen atom or ahydroxy group, or a salt of said compound.

According to the present invention, there is further provided ananti-osteopathic composition comprising as an active ingredient acompound represented by the following formula (II) or (III): ##STR5##wherein R₁₁ and R₁₂ independently represent a hydrogen atom or a loweror middle alkyl group or R₁₁ and R₁₂ may combine to form a 6-memberedcycloalkyl ring together with carbon atoms substituted by these groupsand said cycloalkyl ring may contain an oxygen atom and may besubstituted by one or more lower alkyl groups; R₁₃ represents a hydrogenatom or a lower alkyl group; and W represents a hydrogen atom or ahydroxy group, or a salt of said compound.

Specific examples of the carboxylic acids and their derivativesaccording to the present invention represented by the aforementionedformula (I) include the following compounds, but the present inventionis not limited to these compounds:

4-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carboxamide]benzoicacid (hereinafter referred to as "Am580");

4-[(3,4-diisopropylphenyl)carboxamide]benzoic acid;

5-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carboxamide]pyridine-2-carboxylicacid;

6-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carboxamide]pyridine-3-carboxylicacid;

3-hydroxy-4-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carboxamide]benzoicacid (hereinafter referred to as "Am589");

4-[(3-isopropyl-4-isopropoxyphenyl)carboxamide]benzoic acid (hereinafterreferred to as "Am685");

3-hydroxy-4-[(3-isopropyl-4-isopropoxyphenyl)carboxamide]-benzoic acid(hereinafter referred to as "Am689");

4-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carbamoyl]benzoicacid (hereinafter referred to as "Am80");

4-[(3,4-diisopropylphenyl)carbamoyl]benzoic acid;

5-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carbamoyl]pyridine-2-carboxylicacid;

(E)-4-[3-(3,5-di-tert-butylphenyl)-3-oxo-1-propenyl]benzoic acid(hereinafter referred to as "Ch55");

(E)-4-[3-(3,5-diisopropylphenyl)-3-oxo-1-propenyl]benzoic acid;

6-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carbamoyl]pyridine-3-carboxylicacid (hereinafter referred to as "R300");

4-[3-methyl-3-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)oxylanyl]-benzoicacid;

4-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)azoxy]benzoicacid;

4-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)azo]benzoicacid;

4-[(3,5-di-tert-butylphenyl)carboxamide]benzoic acid;

4-[(3,5-di-tert-butylphenyl)carbamoyl]benzoic acid;

5-[(3,4-diisopropylphenyl)carboxamide]pyridine-2-carboxylic acid;

6-[(3,4-diisopropylphenyl)carboxamide]pyridine-3-carboxylic acid;

5-[(3,4-diisopropylphenyl)carbamoyl]pyridine-2-carboxylic acid;

6-[(3,4-diisopropylphenyl)carbamoyl]pyridine-3-carboxylic acid;

4-[3-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-oxyranyl]benzoicacid (hereinafter referred to as "Ep80"); and

4-[(3,4-diisopropylphenyl)azo]benzoic acid (hereinafter referred to as"Az68").

Examples of the compounds represented by the formula (II) include4-(6,7,8,9-tetrahydro-3-hydroxy-6,6,9,9-tetramethyl-4H-4-oxonaphtho[2.3-b]-pyran-2-yl)benzoicacid (hereinafter referred to as "Fv180") and4-(6,7,8,9-tetrahydro-6,6,9,9-tetramethyl-4H-4-oxonaphtho[2.3-b]pyran-2-yl)-benzoicacid (hereinafter referred to as "Fv80"); and

examples of the compounds represented by the formula (III) include4-[1-hydroxy-3-oxo-3-(5,6,7,8-tetrahydro-3-hydroxy-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoicacid (hereinafter referred to as "Re80"), but the present invention isnot limited to these compounds.

The term "lower or middle alkyl group" herein used means an alkyl grouphaving 1 to 4 carbon atoms such as methyl, ethyl, isopropyl, n-propyl,n-butyl, iso-butyl and tert-butyl, and "lower alkyl" means methyl orethyl group. The term "lower or middle alkoxy group" means an alkoxygroup having 1 to 4 carbon atoms such as methoxy, ethoxy, isopropoxy,n-propoxy, n-butoxy, iso-butoxy and tert-butoxy.

The aforementioned compounds contained in the anti-osteopathiccomposition of the present invention can be prepared, for example,according to methods described in Japanese Patent Unexamined PublicationNos. (Sho) 61-22047, (Sho) 61-76440, (Sho) 62-190154, and (Sho)62-215581, and Japanese Patent Application No. (Hei) 3-328633. However,the methods for preparing the aforementioned compounds are not limitedto these methods. For example, synthetic intermediates for thepreparation of these compounds can be prepared according to a methoddescribed in the Journal of Antibiotic, Vol. 21, No. 10, pp. 603-610,1968.

The anti-osteopathic composition of the present invention comprises, asan active ingredient, the compounds described above in the form of afree compound or a pharmaceutically acceptable salt. Examples of thepharmaceutically acceptable salt include, for example, sodium,potassium, calcium, ammonium and amine salts. The anti-osteopathiccomposition of the present invention may comprise two or more of thecompounds described above in any combination thereof. Furthermore, itmay comprises medicaments for the treatment of osteopathia such as, forexample, vitamin D and calcium and other therapeutically activeingredients.

The aforementioned compounds contained in the anti-osteopathiccomposition of the present invention have potent osteogenesis enhancingactivities. When used as medicaments for the treatment of osteopathia,e.g., osteoporosis, the present compounds exhibit remarkable effects onthe enhancement of osteogenesis. They may also be used for preventingbone fractures, and furthermore, they may be used as medicaments for thetreatment of bone fractures where healing processes necessarily includeosteogenesis.

Route of administration of the anti-osteopathic composition of thepresent invention is not particularly limited, and the composition maybe administered orally or parenterally. For example, pharmaceuticalformulations suitable for oral administration include, for example,tablets, capsules, powders, subtilized granules, granules, liquidcompositions and syrups. Pharmaceutical formulations suitable forparenteral administration include, for example, injections,suppositories, inhalants, ointments, plasters, and implants. Theanti-osteopathic composition of the present invention may be preparedusing pharmacologically and pharmaceutically acceptable additives, ifdesired. Examples of the pharmacologically and pharmaceuticallyacceptable additives include, for example, excipients, disintegratingagents or disintegrators, binders, lubricants, coating agents,colorants, diluents, bases, dissolving agents or solubilizer,isotonizing agents, pH adjusting agents, stabilizers, propellants, andthickening agents.

The pharmaceutical formulations suitable for oral, percutaneous orpermucosal administration can be added with pharmaceutical additivessuch as, for example, an excipient such as glucose, lactose, D-mannitol,starch and crystalline cellulose; a disintegrating agent or adisintegrator such as carboxymethylcellulose, starch, and calciumcarboxymethylcellulose; a binder such as hydroxypropylcellulose,hydroxypropylmethylcellulose, polyvinylpyrrolidone, and gelatin; alubricant such as magnesium stearate, and talc; a coating agent such ashydroxypropylmethyl-cellulose, saccharose, polyethylene glycol, andtitanium oxide; a base such as petrolatum, liquid paraffin, polyethyleneglycol, gelatin, kaolin, glycerine, purified water, and hard fat; apropellant such as flon, diethyl ether and pressured gas; a thickeningagent such as sodium polyacrylate, polyvinyl alcohol, methylcellulose,polyisobutylene and polybutene; and a base sheet such as cotton fabricand plastic sheet. The pharmaceutical formulations suitable forinjection can be added with pharmaceutical additives such as, forexample, a dissolving agent or a solubilizer such as distilled water,physiologic saline, and propylene glycol which can be comprised of anaqueous injections or an injection to be dissolved upon using; anisotonizing agent such as glucose, sodium chloride, D-mannitol, andglycerine; a pH adjusting agent such as inorganic acids, organic acids,inorganic bases, and organic bases. Furthermore, the pharmaceuticalpreparation may be administered as an implant at a site of a bonefracture to enhance an effect of the treatment of the fracture.

The dose of administration of the anti-osteopathic composition of thepresent invention is not particularly limited, and the dose may suitablybe choosen depending on the route of administration, age, body weight,and condition of a patient. For example, where the composition isadministered orally, it can be administered to an adult patient in anamount of 0.05 to 100 mg, preferably, 0.1 to 50 mg per day. Theanti-osteopathic composition of the present invention may beadministered once or several times a day. In addition, a period of timefor administration may also suitably be choosen depending on age,condition of a patient and the like.

The osteogenesis enhancing activities of the compounds contained in theanti-osteopathic composition of the present invention has beendemonstrated by an in vitro experiment in which theiralkaline-phosphatase activities were studied by using an osteoblast-likemouse cell-line MC3T3-E1 (H. Kodama et al., Jpn. J. Oral. Biol., Vol.23, p.899-901, 1981). Efficacy of the compounds in vivo was alsorevealed by examining their activities on rat bone atrophy which wasexperimentally induced by immobilization.

More specifically, during a course of experiments in vitro in whicheffects on an activity of alkaline phosphatase, a marker enzyme ofosteoblast having osteogenetic functions and is generally reported tocontribute to a calcification of calcium, i.e., an activation ofosteogenesis, it was found that the aforementioned compounds activateosteogenesis of osteoblasts. In addition, an animal model was used as anexperimental system in vivo in which bone atrophy was induced byneurectomy in a rat fore-leg to cause immobilization of the fore-leg.This model system has been widely used for developments of medicamentsfor therapeutic treatments of osteopathia including osteoporosis. It wasdemonstrated that the aforementioned compounds exhibit remarkableefficacy in this model system.

EXAMPLES

The present invention will be further explained hereinafter by way ofexamples, but the scope of the present invention is not limited to theseexamples.

Preparation of novel compounds contained in the anti-osteopathiccomposition of the present invention are specifically explained by wayof preparation examples set out below.

PREPARATION EXAMPLE 1

(i) To a mixture of acetic anhydride (37.8 g) and pyridine (40.1 g),o-isopropylphenol (10.7 g) was added at 0° C., and then the mixture wasstirred for 2 hours at room temperature. After a completion of thereaction, the reaction mixture was poured into ice-water, and themixture was extracted with ethyl acetate. The organic layer wasconcentrated under reduced pressure, and the residue was purified bycolumn chromatography using silica gel (n-hexane: ethyl acetate=30:1) togive o-isopropylphenylacetate (13.3 g) as an oil.

(ii) To a mixture of the compound obtained in the above step (i) (13.3g) and nitrobenzene (70 ml), aluminium trichloride (14.0 g) was addedfourfold portionwise, and then the mixture was stirred at roomtemperature for 24 hours. The reaction mixture was poured into anaqueous hydrochloric acid, and the mixture was extracted with ethylacetate. Ethyl acetate and nitrobenzene were evaporated under reducedpressure, and the resulting crystals were washed with n-hexane to give3-isopropyl-4-hydroxyacetophenone (10.0 g).

(iii) A mixture of the compound obtained in the above step (ii) (4.9 g),potassium carbonate (15.1 g), isopropylbromide (6.9 g), and dry acetone(100 ml) was stirred under reflux for 24 hours. After a completion ofthe reaction, the reaction mixture was poured into ice-water, and thenthe mixture was extracted with ethyl acetate. The organic layer wasconcentrated under reduced pressure, and the residue was purified bycolumn chromatography using silica gel (n-hexane: ethyl acetate=3:2) togive 3-isopropyl-4-isopropoxyacetophenone (5.5 g) as an oil.

(iv) A mixture of chlorinated lime (12.5 g), potassium carbonate (8.6g), potassium hydroxide (2.5 g), and water (45 ml) was vigorouslystirred for 30 minutes at 60°-70° C., and then the mixture was filtered.The compound obtained in the above step (iii) (5.5 g) was added to thefiltrate, and then the mixture was stirred for 5 hours at 50°-60° C.After a completion of the reaction, the reaction mixture was extractedwith ether to remove by-products. The aqueous layer was adjusted to pH 1and extracted with ether. The organic layer was concentrated underreduced pressure to give crude crystals. The crystals were washed withn-hexane to afford 3-isopropyl-4-isopropoxybenzoic acid (3.7 g).

(v) To the compound obtained in the above step (iv)(1.8 g), thionylchloride (10 ml) and dimethylformamide (2 drops) were added, and thenthe mixture was stirred at 3 hours at room temperature. The reactionmixture was concentrated to dryness under reduced pressure to remove anexcess thionyl chloride. Pyridine (15 ml), ethyl p-aminobenzoate (1.4g), and dimethylaminopyridine (100 mg) were added to the residue andthen the mixture was stirred for 3 hours at room temperature. After acompletion of the reaction, the reaction mixture was poured intoice-water, and the mixture was extracted with ethyl acetate. The organiclayer was concentrated under reduced pressure, and the resulting residuewas purified by column chromatography using silica gel(chloroform:methanol=30:1) to give an ester compound (2.5 g). Ethanol(20 ml) and 4N aqueous sodium hydroxide solution (8 ml) was added to theester, and the mixture was stirred for 6 hours at about 50° C. After acompletion of the reaction, the reaction mixture was concentrated underreduced pressure. Water was added to the residue and the mixture wasacidified using an aqueous hydrochloric acid to obtain crude crystals.The crystals were washed with a mixture of small volume of ether andn-hexane to give 4-[(3-isopropyl-4-isopropoxyphenyl)carboxamide]benzoicacid (1.9 g).

MS (El): m/z 341 (M⁺), 205, 163

PREPARATION EXAMPLE 2

In the same manner as preparation example1,3-isopropyl-4-isopropoxybenzoic acid was condensed with ethyl4-aminosalicylate and the resulting ester was subjected to ahydrolization to give3-hydroxy-4-[(3-isopropyl-4-isopropoxyphenyl)carboxamide]benzoic acid.

MS (El): m/z 357 (M⁺), 313, 205, 163

Experiment 1: Measurement of activity of alkaline-phosphatase

MC3T3-E1 cells, established cell line of osteoblastic cell, weresubcultured in α-MEM culture medium containing 10% fetal bovine serumplaced in an incubator under 5% CO2 atmosphere at 37° C. The MC3T3-E1cells described above were seeded into each well of a 24-well platecontaining 1 ml of culture medium up to 1·10⁴ cells/well and culturedfor 3 days in the incubator. The cultured medium was removed and then 1ml of α-MEM culture medium containing each test compound atconcentrations of from 10⁻⁵ to 10⁻¹² M and 0.3% bovine serum albumin wasadded and cultured for additional 2 days. Then, the cultured medium wasremoved and the cells were added with 200 μml of 0.2% Nonidet P-40(Nakarai Kagaku Yakuhin Co.) solution containing 1 mM magnesium chlorideand incubated for 1 hour at 37° C. The extract solution was transferredinto a test tube and centrifuged, and then 100 μl of the resultingsupernatant solution obtained by the centrifugation was added to 100 μlof a substrate solution (0.2M glycine buffer (pH 10.4), 20 mM zincchloride, 20 mM magnesium chloride, 20 mM p-nitrophenyl phosphate),which was preliminarily warmed for 3 minutes at 37° C. The mixture wasallowed to react for 120 minutes at 37° C. The reaction was stopped byadding 1.5 ml of 0.15M sodium hydroxide solution and alkalinephosphatase activity was determined by measuring absorbance at 420 nmusing a spectrophotometer.

Minimum effective concentrations of the compounds which inducesignificant increases of alkaline-phosphatase activities as comparedwith a compound-free reference are set out in Table 1. By t-testevaluation of the significant increases, p values of less than 0.01 wereobtained.

                  TABLE 1                                                         ______________________________________                                        Test Compound                                                                             Minimum Effective Concentration (M)                               ______________________________________                                        Etretinate  10.sup.-7                                                         Re 80       .sup. 10.sup.-10                                                  Fv80        .sup. 10.sup.-10                                                  Am580       .sup. 10.sup.-10                                                  Am80        10.sup.-9                                                         R300        10.sup.-9                                                         Az68        10.sup.-9                                                         Ch55        .sup. 10.sup.-10                                                  Ep80        10.sup.-9                                                         Fv180       10.sup.-9                                                         Am685       10.sup.-9                                                         Am589       10.sup.-9                                                         Am689       10.sup.-8                                                         ______________________________________                                    

Chemical structures of the compounds used in the above Experiment 1 areset out below: ##STR6##

It is apparent from the results summarized above that the compounds Re80and Am580 contained in the anti-osteopathic composition of the presentinvention have remarkable effects on osteogenesis of osteoblasts. It isalso apparent that these effects are significantly remarkable ascompared with etretinate and that the compounds contained in theanti-osteopathic composition of the present invention have extremelyhigh activities.

Experiment 2: Effects on a model of bone atrophy by immobilization.

Models of bone atrophy by immobilization were prepared by brachialneurectomy in the left axia of 6-week old male SD rats anesthetized withpentobarbital. After two weeks from the neurectomy, a suspension ofretinoic acid, etretinate or Am580 contained in the anti-osteopathiccomposition of the present invention in a solution of 0.5% CMC wasadministered orally to a group of rat consisting of 6 treated rats oncea day for two weeks at a daily dose of 0.01 mg/kg or 0.1 mg/kg. The samevolume of 0.5% CMC solution was administered in a similar manner to asolvent-administered reference group. After completion of successiveadministration for 2 weeks, left radius (the side of neurectomy) andright radius (the side of non-neurectomy) were removed. After analcoholic dehydration and defatting, the bones were dried at 160° C. for6 hours to measure their dry weights. The bones were then heated forashing at 600° C. for 24 hours to measure the weights of ash content. Asa non-treated reference group, left and right radiuses and the boneswere treated in the same manner to measure bone dry weight and ashcontent. Efficacy of each compound was evaluated by t-test between themeasurements of the group administered with the test compound and thoseof the two reference groups. The results are summarized in Table 2.

                                      TABLE 2                                     __________________________________________________________________________           Dry Weight of Radius (mg)                                                                     Ash Weight of Radius (mg)                              Compound                                                                             Left.sup.1                                                                            Right.sup.2                                                                           Left.sup.1                                                                            Right.sup.2                                    __________________________________________________________________________    Ref..sup.3                                                                           109.3 ± 5.3                                                                        107.7 ± 2.6                                                                        76.5 ± 3.8                                                                         75.1 ± 2.3                                         (100)   (100)   (100)   (100)                                          Ref..sup.4                                                                           94.1 ± 1.8A                                                                        108.3 ± 2.0                                                                        64.9 ± 1.0A                                                                        75.1 ± 1.4                                         (86.1)  (100.6) (84.8)  (100.0)                                        Am580  99.5 ± 6.2a                                                                        111.2 ± 6.0                                                                        69.3 ± 5.0a                                                                        77.0 ± 4.3                                  (0.01 mg/kg)                                                                         (91.0)  (103.2) (90.6)  (102.5)                                        Am580  101.5 ± 4.1a,b                                                                     118.2 ± 3.5A,B                                                                     70.1 ± 2.3A,B                                                                      82.1 ± 2.4A,B                               (0.1 mg/kg)                                                                          (92.9)  (109.7) (91.6)  (109.3)                                        Ref..sup.3                                                                           118.3 ± 6.2                                                                        118.1 ± 4.8                                                                        82.7 ± 3.4                                                                         82.5 ± 3.1                                         (100)   (100)   (100)   (100)                                          Ref..sup.4                                                                           105.2 ± 4.4A                                                                       121.4 ± 2.8                                                                        72.9 ± 3.0A                                                                        84.5 ± 2.1                                         (88.9)  (102.8) (88.1)  (102.4)                                        Retinoic Acid                                                                        100.1 ± 4.7A                                                                       114.9 ± 6.3b                                                                       69.0 ± 3.0Ab                                                                       80.1 ± 4.4                                  (0.01 mg/kg)                                                                         (84.6)  (97.3)  (83.4)  (97.1)                                         Retinoic Acid                                                                        100.6 ± 4.2A                                                                       122.2 ± 4.1                                                                        73.4 ± 2.7A                                                                        84.8 ± 2.4                                  (0.1 mg/kg)                                                                          (85.0)  (103.5) (88.8)  (102.8)                                        Ref..sup.3                                                                           112.6 ± 3.7                                                                        111.6 ± 3.6                                                                        80.3 ± 2.6                                                                         79.6 ± 2.8                                         (100)   (100)   (100)   (100)                                          Ref..sup.4                                                                           97.7 ± 12.8a                                                                       111.0 ± 11.6                                                                       68.6 ± 9.1a                                                                        78.1 ± 7.8                                         (86.8)  (99.5)  (85.4)  (98.1)                                         Etretinate                                                                           97.8 ± 4.7A                                                                        113.6 ± 7.8                                                                        68.1 ± 3.5A                                                                        79.6 ± 5.6                                  (0.01 mg/kg)                                                                         (86.9)  (101.8) (84.8)  (100.0)                                        Etretinate                                                                           100.9 ± 2.4A                                                                       115.9 ± 4.3                                                                        69.8 ± 1.6A                                                                        80.6 ± 2.8                                  (0.1 mg/kg)                                                                          (89.6)  (103.9) (86.9)  (101.3)                                        __________________________________________________________________________     .sup.1 The side of neurectomy                                                 .sup.2 The side of nonneurectomy                                              .sup.3 Nontreated Reference                                                   .sup.4 Solventadministered Reference                                          A,a, A significant difference was observed in comparison with nontreating     reference.                                                                    a = p < 0.05, A = p < 0.01                                                    B,b, A significant difference was observed in comparison with                 solventadministered reference.                                                b = p < 0.05, B = p < 0.01                                                    () Relative value where the values of nontreating reference equals to 100                                                                              

By brachial neurectomy, the dry weights and ash weight of the radius inthe side of neurectomy were significantly decreased. However, nodecrease was found in the side of non-neurectomy, and accordingly, noeffect of the neurectomy was observed in the side of non-neurectomy. Itwas found that Am580, one of the compounds contained in theanti-osteopathic composition of the present invention, has potency ofinhibitory effect on decreases of dry weight and ash weight of radius atthe dose of 0.01 mg/kg, although the differences were insignificant, andit remarkably inhibits the decrease at the dose of 0.1 mg/kg. In theside of non-neurectomy, significant increases of dry weights and ashweights of radiuses were observed in the group administered with 0.1mg/kg of Am580 as compared to non-treated and solvent administeredreference groups, which is evidence of an enhancing activity onosteogenesis. In the group administered with etretinate, it was revealedthat etretinate has a potency of inhibiting the decrease of dry weightand ash weight of the radius in the side of neurectomy and it also has apotency of inducing the weight increase of the bones in the side ofnon-neurectomy at the dose of 0.1 mg/kg, although the differences wereinsignificant. Retinoic acid did not have significant effect in thisexperimental system.

The above-described results clearly show that Am580, one of thecompounds contained in the anti-osteopathic composition of the presentinvention, exhibits a remarkable inhibitory activity on the decrease ofbone amount and an enhancing activity on osteogenesis. The activitiesare unexpectedly remarkable as compared with those of retinoic acid andetretinate, and therefore, it is apparent that the anti-osteopathiccomposition according to the present invention is useful.

We claim:
 1. A method for treatment of a bone disease which comprises astep of administering to a patient in recognized need thereof, ananti-osteopathic composition comprising as an active ingredient acompound represented by the following formula (I): ##STR7## wherein R₁,R₄, and R₅ are hydrogen atoms; R₂ and R₃ independently represent a C₁-C₄ alkyl group or a C₁ -C₄ alkoxy group or R₂ and R₃ may combine toform a 5- or 6-membered cycloalkyl ring together with carbon atoms towhich they are bonded, wherein the cycloalkyl ring can be substitutedwith one or more C₁ -C₂ alkyl groups; R₆ represents hydroxy or a C₁ -C₂alkoxy group; X is selected from the group consisting of --NH--CO-- and--CO--NH--; and Y is selected from the group consisting of: ##STR8##wherein W is hydrogen or hydroxy, or a pharmaceutically acceptable saltthereof.